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Feb 17, 2017 - Quasar Energy Group, 2705 Selby Road, Wooster, Ohio 44691, United States. ⊥ Engineering Research Center of Industrial Microbiology, ...
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Methanol Production from Biogas with a Thermotolerant Methanotrophic Consortium Isolated from an Anaerobic Digestion System Zhongliang Su,†,‡,§ Xumeng Ge,*,†,‡,∥ Wenxian Zhang,‡,⊥ Lingling Wang,# Zhongtang Yu,# and Yebo Li*,‡ ‡

Department of Food, Agricultural and Biological Engineering, The Ohio State University/Ohio Agricultural Research and Development Center, 1680 Madison Avenue, Wooster, Ohio 44691-4096, United States § Department of Biotechnology, Qingdao University of Science and Technology, Post Office Box 47, 53 Zhengzhou Road, Qingdao, Shandong 266042, People’s Republic of China ∥ Quasar Energy Group, 2705 Selby Road, Wooster, Ohio 44691, United States ⊥ Engineering Research Center of Industrial Microbiology, Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, Fujian 350108, People’s Republic of China # Department of Animal Sciences, The Ohio State University, 2029 Fyffe Court, Columbus, Ohio 43210, United States ABSTRACT: Thermotolerant methanotrophic consortia are desirable for their robustness under stressful environments during industrial applications. A thermotolerant methanotrophic consortium, MC-AD3, was enriched from digestate in an anaerobic digestion (AD) system and evaluated for cell growth and methanol production with biogas. MC-AD3 obtained cell yields of 0.22−0.40 g of cells/g of methane at temperatures from 30 to 55 °C and pH from 5.5 to 7.5 and achieved the highest cell yield of 0.4 g of cells/g of methane at 47 °C at a biogas/air ratio of 1:4 (v/v) and pH of 6.8. MC-AD3 produced 0.33 g/L of methanol at 47 °C, with a methanol conversion ratio of 0.47 mol of methanol/mol of methane. A biogas/air ratio of 1:1 (v/v) was found to be optimal for methanol production with MC-AD3. The cell growth and methanol production performance of MC-AD3 at 47 °C fell within the range of those obtained by other methanotrophic strains/consortia at lower temperatures.

1. INTRODUCTION Biogas (mainly methane and carbon dioxide) is produced from anaerobic digestion (AD) of organic wastes, such as crop residues, municipal solid waste, food waste, sewage sludge, and animal manure.1,2 As a renewable source of energy, biogas has been used to produce electricity using combined heat and power (CHP) systems.3 Nevertheless, biogas is in a gaseous form at ambient temperatures and is difficult and costly to store, transport, and distribute.4 This issue can be addressed by conversion of methane to methanol, which is an important building block for transportation fuels and chemicals.5 Methane-to-methanol conversion is commonly conducted via thermochemical conversion, which involves high pressure and/ or temperature and expensive chemical catalysts.6 In comparison to thermochemical conversion methods, biological conversion of biogas to methanol is more attractive for biogas upgrading as a result of efficient conversion reactions under mild conditions.7−9 Methanotrophs are a group of bacteria that can use methane as the only carbon source for growth.4 Currently, all enriched/ isolated methanotrophs are aerobic. Although anaerobic methanotrophs (ANME) have also been identified, no ANME have been isolated in either pure culture or a consortium up to date.4 Methanotrophs generally convert methane to methanol with methane monooxygenase (MMO), consuming two reducing equivalents per molecule of methane oxidized.1,10 Under normal conditions, methanotrophs further oxidize methanol to CO2 with formaldehyde and formate as © 2017 American Chemical Society

intermediates, which are catalyzed by methanol dehydrogenase (MDH), formaldehyde dehydrogenase (FalDH), and formate dehydrogenase (FDH), in a sequential manner.1,10 Methanol accumulation is generally conducted with the addition of MDH inhibitors, including phosphate, ethylenediaminetetraacetic acid (EDTA), MgCl2, high salinity, and cyclopropanol.4,11,12 When MDH is inhibited, external electron donors, mainly formate, are needed to maintain cell vitality of methanotrophs, which can only use monocarbon compounds.4 Production of formic acid from CO2 and H2O has been achieved via electrochemical methods, which offer a cheap supply of formate as an electron donor.13,14 However, studies on biological conversion of biogas to methanol are still limited, and there are challenges for industrial-scale production.4 One of these challenges is the lack of robust methanotrophic strains with high tolerance to environmental stresses that may occur during industrial applications.4,15 Pure methanotrophic strains, mainly Methylosinus trichosporium, are routinely used for methanol production from methane at a lab scale.4,16 However, pure methanotrophs are generally vulnerable to contamination by other microorganisms, posing a high risk in industrial-scale applications. It is believed that diversity in bacterial communities can promote stability of ecosystems.17 For Received: December 27, 2016 Revised: February 16, 2017 Published: February 17, 2017 2970

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Energy & Fuels Table 1. Selection of Methanotrophic Consortia Isolated from Digestates from Different AD Systems AD condition number C1 C2 AD1 AD2 AD3 AD4 AD5 AD6 a

feedstock

TS (%)a

F/Ib

T (°C)

T (°C) for enrichment

cell growth

methanol production (mg/L)

37 55 37 55 37 37 37 37

+ − + − + − + +

192

c

expired dog food expired dog food corn stover corn stover giant reed giant reed

20 20 20 20 20 8

2 2 3 4 2 1

37 55c 37 55 37 37 37 37

213 276 147 205

Total solids content. bFeedstock/inoculum ratio. cAD effluent was activated at the temperature for 1 week. (0.272 g L−1), Na2HPO4 (0.284 g L−1), CaCl2·2H2O (0.134 g L−1), chelated Fe solution (0.2%, v/v), and a trace element solution (0.05%, v/v). The chelated Fe solution was prepared by dissolving ferric (III) ammonium citrate (1.0 g L−1), EDTA (2.0 g L−1), and concentrated HCl (0.3%, v/v) in deionized (DI) water. The trace element solution was prepared by dissolving EDTA (500 mg L−1), FeSO4·7H2O (200 mg L−1), ZnSO4·7H2O (10 mg L−1), MnCl2·4H2O (3.0 mg L−1), H3BO3 (30 mg L−1), CoCl2·6H2O (20 mg L−1), CaCl2·2H2O (1.0 mg L−1), NiCl2·6H2O (2.0 mg L−1), and Na2MoO4·2H2O (3.0 mg L−1) in DI water. A total of 5 mL of each suspension was inoculated into 50 mL of NMS medium in a 250 mL flask. The flask was filled with 20% (v/v) CH4 (99% purity, purchased from Praxair, Danbury, CT, U.S.A.) and 80% (v/v) air in its headspace and sealed with a rubber stopper. The flasks were incubated at 37 or 55 °C and stirred at 150 rpm for enrichment of methanotrophs (Table 1). Every 5 days, 5 mL of the culture was successively transferred to 50 mL of fresh NMS medium in another 250 mL flask, which was then filled with the gas mixture (1:4, v/v, CH4/air) and incubated under the same conditions. The enrichment process was repeated 3 times. Consortia were cultured in 2 mL of NMS medium supplemented with 2 μM CuCl2 and 50 mM sodium formate in 15 mL sealed culture tubes. The tubes were filled with CH4 and air (1:4, v/v) in their headspace, incubated at 37 °C, and stirred at 150 rpm for 48 h. Cell suspensions were filtered (0.2 μm), and the filtrates were subjected to methanol analysis using gas chromatography (GC). The methanotrophic consortium from AD3 (MC-AD3), which obtained the highest methanol concentration, was selected for further evaluation. The microbial community in MC-AD3 was analyzed with 16S rRNA gene sequencing. It was revealed that MC-AD3 contained methanotrophic bacterium Methylocaldum (87.21%) and other bacteria, including Agrobacterium (7.02%), Alcaligenaceae (4.66%), Stenotrophomonas (0.42%), Limnohabitans (0.11%), and Paenibacillus (0.05%). 2.2. Cell Growth on Biogas under Different Conditions. Biogas samples (A, 72.04% CH4, 26.02% CO2, 1.66% N2, and 0.28% O2; B, 64.09% CH4, 34.39% CO2, 0.97% N2, and 0.55% O2) were collected from a commercial-scale digester (Quasar Energy Group, Wooster, OH, U.S.A.), which was fed with food waste.26 MC-AD3 was inoculated into 30 mL of NMS medium in 250 mL flasks to reach an initial cell density of about 0.4 g/L. Each flask was connected to a 500 mL Tedlar gas bag, and the headspace of the flask and Tedlar gas bag was filled with a specified biogas/air mixture with a total gas volume of 500 mL. The flasks were inoculated at the designated temperature and shaken at 150 rpm for 192 h. Gas composition (CH4, O2, N2, and CO2) was monitored every day. Cell density was measured after the cultivation for determination of the cell yield from methane (grams of dry biomass produced per gram of methane consumed). The effect of the biogas/air ratio, temperature, and pH on cell growth of MC-AD3 was evaluated in a sequential manner. Briefly, the optimal biogas/air ratio was first determined with cell cultivation at 37 °C and a pH of 6.8 with different biogas/air ratios (1:2, 1:4, and 1:6, v/v) according to a previous study.25 The second run of cell cultivation was then conducted at the optimal biogas/air ratio, a pH of 6.8, but different temperatures (30, 37, 42, 47, 50, and 55 °C), to determine

example, researchers in microalgae-based biofuel production have proposed to use wild consortia of microalgae rather than monospecific microalgal cultures to reduce the risk of contamination by “weed” microalgae or predators.18 Similar strategies might be effective for methanol production with methanotrophs. However, only one study has reported methanol production from biogas with a methanotrophic consortium isolated from landfill cover soil.19 Another strategy to minimize contamination by other microorganisms is to use thermophilic or thermotolerant strains that can grow at relatively high temperatures.20 Besides, thermotolerant strains are also desirable for reduced cooling requirement, which could be challenging for a large-scale fermentation process, during which heat is generally removed using a cooling device.21 Several thermophilic and thermotolerant methanotrophs have been identified with optimum growth at 37−57 °C, although they have not been used for methanol production.22 Isolation of robust methanotrophic consortia is a promising option for methanol production from biogas. To date, there have been no reports on the isolation of thermophilic/thermotolerant methanotrophic consortia for methanol production from biogas. The objectives of this study were to (1) isolate thermophilic/ thermotolerant methanotrophic consortia from digestate in AD systems and (2) evaluate environmental conditions on cell growth and methanol production with selected consortia. Digestates from different AD systems were used as sources for isolation of methanotrophic consortia. One thermotolerant methanotrophic consortium that accumulated higher methanol than others was selected. The effects of the biogas/air ratio, temperature, and/or pH on cell growth and methanol production with this consortium were investigated.

2. MATERIALS AND METHODS 2.1. Enrichment of Methanotrophic Consortia from Digestate. Enrichment of methantrophic consortia was conducted on the digestate from AD experiments that used different feedstocks (expired dog food, corn stover, and giant reed) with different total solids (TS) contents and feedstock/inoculum (F/I) ratios. These AD experiments were conducted for previous studies, and specific conditions for each are shown in Table 1. The AD effluent collected from a mesophilic liquid anaerobic digester (KB BioEnergy, Akron, OH, U.S.A.) was activated at 37 or 55 °C for 1 week and used as an inoculum for AD. The activated AD effluent and digestate collected after AD experiments were used as sources prior to use for enrichment. The enrichment was conducted according to protocols reported by Bowman,23 Dedysh and Dunfield,24 and Sheets et al.25 Briefly, 5 g of digestate sample was mixed with 20 mL of nitrate mineral salts (NMS) medium in a 125 mL flask (in duplicate). The NMS medium contained MgSO4·7H2O (1.0 g L−1), KNO3 (1.0 g L−1), KH2PO4 2971

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2.5. Statistical Analysis. Analysis of variance (one-way ANOVA; α = 0.05; i.e., confidence level = 95%) was conducted using Minitab (version 16, Minitab, Inc., State College, PA, U.S.A.) for assessing statistical significance.

the optimal temperature. Optimal pH was determined with the third run of cell cultivation at the optimal biogas/air ratio and temperature with different pH levels (5.5, 6.0, 6.8, and 7.5). Three replicates were used for each condition. Biogas sample A was collected first and used for the experiment of the effect of the biogas/air ratio on cell growth. To allow for more repeated trails for further experiments, biogas sample B was collected with a larger volume. After the experiment on the optimization of the temperature for cell growth, the gas bag of sample B was accidentally broken. As a result, biogas sample A was used for all of the rest of experiments. 2.3. Methanol Production from Biogas under Different Conditions. MC-AD3 was grown in 30 mL of NMS medium in 250 mL flasks at 37 °C and a pH of 6.8 with a biogas/air (1:4, v/v) mixture in the headspace. Four flasks were setup for each condition. After 8 days, MC-AD3 cultures in the four flasks were combined, and the cells were harvested by centrifugation at 8000g for 10 min. The harvested cells were resuspended in 120 mL of fresh NMS medium (pH 6.8) with 5 μM CuCl2 and 100 mM sodium formate. The cell suspension was mixed well and transferred into four 250 mL flasks (30 mL in each flask). One of the flasks was used to determine initial cell density (about 0.23 g/L, dry weight). The other three were used for methanol production. Each of the three flasks was connected to a 500 mL Tedlar gas bag. The headspace of the flask and Tedlar gas bag was filled with a biogas/air (1:4, v/v) mixture to reach a total volume of 700 mL, and the flask was inoculated at a designated temperature with shaking at 150 rpm for 168 h. Gas composition (CH4, O2, N2, and CO2) and methanol concentration were monitored every 12−24 h. The methanol yield from methane (moles of methanol produced per mole of methane consumed) was calculated to determine optimal conditions. Methanol production with MC-AD3 was conducted at a biogas/air ratio of 1:1 (v/v) and different temperatures (37, 47, and 55 °C), to evaluate the effect of the temperature on methanol production. Effects of the biogas/air ratio on methanol production were further evaluated for methanol production at 47 °C with different biogas/air ratios (2:1, 1:1, 1:2, 1:4, and 1:6, v/v). Three replicates were used for each condition. 2.4. Analytical Methods. Cell density was determined using a method originated by Zhu and Lee27 and modified by Sheets et al.25 Briefly, 30 mL of cell suspension was centrifuged at 10 000 rpm for 15 min, and the supernatant was discarded. To remove residual salts, the cell pellet was resuspended in 25 mL of 0.5 M NH4HCO3 and centrifuged again at 10 000 rpm for 15 min. After the supernatant was discarded, the cell pellet was transferred to a pre-ignited (550 °C) porcelain crucible with 3 mL of 0.5 M NH4HCO3 and dried in a Thelco Model 18 oven (Precision Scientific, Chennai, India) at 105 °C for 12 h to determine the dry weight of total biomass. The dry biomass sample was heated in an Isotemp muffle furnace (Fisher Scientific, Dubuque, IA, U.S.A.) at 550 °C for 4 h to determine the ash weight. The ash-free dry weight was calculated as the difference between the dry weight of total biomass and the weight of the residual ash. Gas composition (CH4, CO2, N2, and O2) was analyzed using a gas chromatograph (Agilent, HP 6890, Wilmington, DE, U.S.A.), which was equipped with a 30 m × 0.53 mm × 10 μm Rt-Alumina BOND/ KCl deactivation column and a thermal conductivity detector. Helium gas was used as the carrier gas with a flow rate of 5.2 mL/min. Temperatures of the injector and detector were set at 150 and 200 °C, respectively. The temperature of the column oven was initially set at 40 °C for 4 min, later increased to 60 °C at 20 °C/min, and held at 60 °C for 5 min. The methanol concentration in the filtrate samples was analyzed using a gas chromatograph (Shimadzu, 2010PLUS, Columbia, MD, U.S.A.), which was equipped with a Stabilwax polar phase column (30 m × 0.32 mm × 0.5 μm) and a flame ionization detector. The temperatures of both the injector and detector were set at 250 °C, while the temperature of the column oven was initially set at 50 °C and gradually increased to 80 °C at a rate of 5.0 °C/min. Helium was used as the carrier gas with a total flow rate of 24.8 mL/min and a split ratio of 15.

3. RESULTS AND DISCUSSION 3.1. Enrichment of Methanotrophic Consortia from Digestate. Generally, methanotrophic consortia were obtained from digestate in mesophilic AD systems via enrichment at 37 °C (Table 1). One exception was that enrichment of methanotrophic consortia failed using digestate from AD4 (Table 1). AD4 was conducted at a high F/I ratio and was upset with a low pH of about 5 as a result of volatile fatty acid accumulation (data not shown). Methanotrophs in the AD system might not tolerate the low pH, which could have resulted in the failure of enrichment. Besides, no methanotrophic consortia were enriched at 55 °C. These results indicated that the AD systems as well as the inoculum (AD effluent) likely lack acidophilic and thermophilic methanotrophs (Table 1). All of the enriched methanotrophic consortia produced methanol. The highest methanol concentration (276 mg/L) was obtained with the methanotrophic consortium enriched from digestate in AD3 (Table 1). This consortium was named as MC-AD3 and used for further experiments. 3.2. Effect of the Biogas/Air Ratio on Cell Growth of MC-AD3. When MC-AD3 was cultivated at a biogas/air ratio of 1:2 (v/v), the methane content decreased slowly from 24 to 18% in 8 days (Figure 1a). A biogas/air ratio of 1:4 (v/v)

Figure 1. Effect of the biogas/air ratio on (a) methane consumption and (b) cell yield from methane during cultivation of MC-AD3 using biogas as the carbon source at 37 °C and an initial pH of 6.8. Means that do not share a letter are significantly different.

resulted in a faster decrease (from 15 to 4%) of methane content than a biogas/air ratio of 1:2 (v/v) during 8 days of cell growth (Figure 1a). However, when the biogas/air ratio was decreased to 1:6 (v/v), the methane content decreased from 10 to 1% in 8 days, showing a slightly slower decrease of the methane content than that obtained with a biogas/air ratio of 1:4 (v/v) (Figure 1a). The cell yields from methane were 0.133, 0.234, and 0.202 g of cells/g of methane when biogas/air ratios were 1:2, 1:4, and 1:6 (v/v), respectively (Figure 1b). The biogas/air ratio of 1:4 (v/v) obtained the fastest decrease of methane content and highest cell yield from methane. However, there was no significant (p > 0.05) difference in the cell yield between biogas/air ratios of 1:4 and 1:6 (v/v) or initial methane contents of 15 and 10% (Figure 1b). This also supports the feasibility of using biogas sample B for further experiments (as mentioned in section 2.2) with an initial methane content of about 12−13% at the biogas/air ratio of 1:4 2972

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Energy & Fuels (v/v). The cell yield from methane with MC-AD3 was comparable to that of Methylocaldum 14B (0.2 g of cells/g of methane), a pure methanotrophic strain that was isolated from solid-state anaerobic digestate.25 However, the cell yields of MC-AD3 were still lower than those (0.5−0.7 g of cells/g of methane) of M. trichosporium OB3b, which indicates that MCAD3 oxidized more methane for energy generation than for biomass production compared to M. trichosporium OB3b.28,29 3.3. Effect of the Temperature and pH on Cell Growth of MC-AD3. Figure 2a shows methane content changes during

Figure 3. Effect of initial pH on (a) methane consumption and (b) cell yield from methane during cultivation of MC-AD3 using biogas as the carbon source at 47 °C and a biogas/air ratio of 1:4 (v/v). There are no significant differences among means.

comparable to that of 14B and SAD2 and to thermophilic/ thermotolerant methanotrophic strains, such as Methylococcus capsulatus, Methylocaldum szegediense, Methylothermus thermalis, and Methylocystis sp. Se48.22,25,30 3.4. Effect of Culture Conditions on Methanol Production from Biogas with MC-AD3. Figure 4 shows Figure 2. Effect of the temperature on (a) methane consumption and (b) cell yield from methane during cultivation of MC-AD3 using biogas as the carbon source with a biogas/air ratio of 1:4 (v/v) and an initial pH of 6.8. Means that do not share a letter are significantly different.

cultivation of MC-AD3 at different temperatures. Interestingly, methane consumption increased as the temperature increased from 30 to 50 °C, although methane consumption decreased when the temperature was further increased to 55 °C (Figure 2a). For example, the methane content decreased from 12 to 3% in 144 h at 37 °C, while the same decrease of the methane content only took 48 h at 50 °C (Figure 2a). Cell yields from methane ranged from 0.22 to 0.40 g of cells/g of methane with temperatures from 30 to 55 °C (Figure 2b). Two methanotrophic strains, 14B and SAD2, which were also isolated from AD systems, grew effectively at 37−42 and 30−37 °C, respectively.25,30 In comparison to methanotrophic strains 14B and SAD2, MC-AD3 had a much wider temperature range for cell growth. Furthermore, the highest cell yield (0.4 g of cells/g of methane) of MC-AD3 was obtained at 47 °C, which indicates that MC-AD3 is a thermotolerant consortium (Figure 2b). According to Trotsenko et al.,22 a few thermophilic and thermotolerant methanotrophs were isolated from sources, such as bottom deposits of water bodies, activated sludge, soil, thermal spring silage, and manure. Most of these methanotrophs had optimum growth at 37−42 °C, and only two showed optimum growth at 55−57 °C.22 This study is the first time that a thermotolerant methanotrophic consortium was enriched from digestate in AD systems. Figure 3 further illustrates the effect of pH on methane consumption and cell yield of MC-AD3 at 47 °C. When the initial pH was 5.0, there was a minimal decrease of the methane content during 6 days, indicating an inhibition of methane consumption with MC-AD3 (Figure 3a). Methane consumption was found to be consistent for initial pH levels of 5.5−7.5, with the methane content decreasing from 15 to 1.3−2.8% in 114 h (Figure 3a). In addition, cell yields from methane consumption were 0.20−0.33 g of cells/g of methane at pH levels from 5.0 to 7.5 without significant (p > 0.05) difference (Figure 3b). The pH range (5.5−7.5) for cell growth was

Figure 4. Effect of the temperature on (a) methanol accumulation and (b) methane/methanol conversion ratio by MC-AD3 using biogas as the carbon source at a biogas/air ratio of 1:1 (v/v). Means that do not share a letter are significantly different.

methanol production performance of MC-AD3 at different temperatures. Interestingly, methanol production with MCAD3 also preferred a relatively high temperature (47 °C) (Figure 4). A methanol concentration of 0.33 g/L was obtained by MC-AD3 at 47 °C, which was 38% higher than that obtained at 37 °C (Figure 4a). MC-AD3 accumulated a minimal amount of methanol (0.04 g/L) at 55 °C (Figure 4a), although it grew stably at this high temperature (Figure 2). The methane/methanol conversion ratio (0.47 mol of methanol/ mol of methane) at 47 °C was also significantly (p < 0.05) higher than those at 37 and 55 °C (Figure 4b). According to Figures 2 and 4, MC-AD3 appeared to be more sensitive to a high temperature for methanol production than for cell growth. Methanol production with methanotrophs has routinely been conducted at 25−37 °C.4,16,25,30 Before this study, there have been no reports on methanol production with methanotrophs at temperatures higher than 37 °C, although thermophilic/ thermotolerant methanotrophs have been isolated and characterized in terms of cell growth.22 As shown in Figure 5a, different biogas/air ratios also resulted in different methanol concentrations during 7 days of the methanol production process. Relatively high methanol concentrations of 0.33 and 0.35 g/L were obtained with biogas/ air ratios of 1:1 (v/v) and 1:2 (v/v), respectively (Figure 5a). 2973

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: +1-330-202-3561. Fax: +1-330-263-3670. E-mail: [email protected]. *Telephone: +1-330-202-3561. Fax: +1-330-263-3670. E-mail: [email protected]. ORCID

Xumeng Ge: 0000-0001-6272-8847 Author Contributions †

Zhongliang Su and Xumeng Ge contributed equally to this work.

Figure 5. Effect of the biogas/air ratio on (a) methanol accumulation and (b) methane/methanol conversion ratio by MC-AD3 using biogas as the carbon source at 47 °C. Means that do not share a letter are significantly different.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This material is based on work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under Award 2012-10008-20302, and the state and federal funds appropriated to The Ohio State University, Ohio Agricultural Research and Development Center. This research is also funded by the China Scholarship Council. The authors thank Mary Wicks for her comprehensive review and thoughtful comments.

The methanol conversion ratio (0.47 mol of methanol/mol of methane) achieved using a biogas/air ratio of 1:1 (v/v) was significantly higher than those obtained with other biogas/air ratios (Figure 5b). According to Figures 1 and 5, MC-AD3 preferred a greater methane content for methanol production than that for cell growth. As show in Table 2, methanol concentrations obtained by MC-AD3 at 47 °C were comparable to those achieved by methanotrophic strains 14B and SAD2, which were also isolated from anaerobic digestate but grew and produced methanol at lower temperatures (around 37 °C) (Table 2).25,30 Besides, the methanol conversion ratio achieved by MC-AD3 at 47 °C falls within the range of those (0.23−0.80 mol of methanol/mol of methane) obtained by other methanotrophic strains/consortia at lower temperatures (25−37 °C) (Table 2). Currently, methanol concentrations obtained by methanotrophic bacteria are still low for large-scale production.4 A major reason is that methanol is toxic to cells at high concentrations, which limits the final methanol concentration.25 Besides directly screening methanol-tolerant methanotrophic strains, it is also promising to investigate genes responsible for the methanol tolerance and further improve the methanol tolerance via genetic manipulations.

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REFERENCES

(1) Strong, P. J.; Kalyuzhnaya, M.; Silverman, J.; Clarke, W. P. Bioresour. Technol. 2016, 215, 314−323. (2) Ge, X.; Xu, F.; Li, Y. Bioresour. Technol. 2016, 205, 239−249. (3) Deublein, D.; Steinhauser, A. In Biogas from Waste and Renewable Resources: An Introduction; Deublein, D., Steinhauser, A., Eds.; WileyVCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2010; pp 477−507, DOI: 10.1002/9783527632794.ch48. (4) Ge, X.; Yang, L.; Sheets, J. P.; Yu, Z.; Li, Y. Biotechnol. Adv. 2014, 32 (8), 1460−1475. (5) Olah, G. A. Angew. Chem., Int. Ed. 2005, 44, 2636−2639. (6) Park, D.; Lee, J. Korean J. Chem. Eng. 2013, 30 (5), 977−987. (7) Yang, L.; Ge, X.; Wan, C.; Yu, F.; Li, Y. Renewable Sustainable Energy Rev. 2014, 40, 1133−1152. (8) Pen, N.; Soussan, L.; Belleville, M.-P.; Sanchez, J.; Charmette, C.; Paolucci-Jeanjean, D. Bioresour. Technol. 2014, 174, 42−52. (9) Strong, P. J.; Xie, S.; Clarke, W. P. Environ. Sci. Technol. 2015, 49, 4001−4018. (10) Hanson, R. S.; Hanson, T. E. Microbiol. Rev. 1996, 60 (2), 439− 471. (11) Hwang, I. Y.; Hur, D. H.; Lee, J. H.; Park, C.; Chang, I. S.; Lee, J. W.; Lee, E. Y. J. Microbiol. Biotechnol. 2015, 25 (3), 375−380. (12) Duan, C.; Luo, M.; Xing, X. Bioresour. Technol. 2011, 102 (15), 7349−7353. (13) Li, H.; Opgenorth, P. H.; Wernick, D. G.; Rogers, S.; Wu, T.-Y.; Higashide, W.; Malati, P.; Huo, Y.-X.; Cho, K. M.; Liao, J. C. Science 2012, 335, 1596. (14) Reda, T.; Plugge, C. M.; Abram, N. J.; Hirst, J. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (31), 10654−10658. (15) Cáceres, M.; Gentina, J. C.; Aroca, G. Biotechnol. Lett. 2014, 36 (1), 69−74.

4. CONCLUSION The thermotolerant methanotrophic consortium, MC-AD3, was isolated from digestate in AD systems. MC-AD3 grew stably for a wide range of temperatures (30−55 °C) and pH values (5.5−7.5) and achieved the highest cell yield (0.4 g of cells/g of methane) at 47 °C with a biogas/air ratio of 1:4 (v/v) and pH of 6.8. MC-AD3 produced 0.33 g/L of methanol at 47 °C with a methanol conversion ratio (0.47 mol of methanol/ mol of methane) that fell within the range of those obtained by other strains at lower temperatures. As a result, MC-AD3 is a promising candidate for conversion of biogas to methanol for large-scale production.

Table 2. Methanol Production Conditions and Performance of Methanotrophic Strains and Consortia strain/consortia

source

T (°C)

methanol concentration (g/L)

CH4/methanol conversion ratio

reference

consortium M. trichosporium strain 14B strain SAD2 MC-AD3

landfill cover soil not reported anaerobic digestate anaerobic digestate anaerobic digestate

30 25−35 37 37 47

0.19−0.22 0.17−1.12 0.43 0.34 0.33

0.43−0.80 0.27−0.61 0.26 0.34 0.47

19 12, 31, and 32 25 30 this study

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DOI: 10.1021/acs.energyfuels.6b03471 Energy Fuels 2017, 31, 2970−2975

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DOI: 10.1021/acs.energyfuels.6b03471 Energy Fuels 2017, 31, 2970−2975